Successful infections by bacterial pathogens require attachment to and colonization of host tissues. Surface determinants (fimbrial and non-fimbrial surface proteins) are essential for these processes in all bacterial pathogens as they provide specific receptor-ligand interactions with tissue factors that determine both bacterial host range and sites of infection. Among the first bacteria to colonize human teeth, actinomyces together with oral streptococci may serve as a foundation for the colonization of other species that are associated with the etiology of carries and periodontal diseases. Fimbriae and non-fimbrial surface components of Actinomyces may have important roles in pathogenesis owing to their ability to interact with and activate host cells. The underlying mechanisms of these interactions remain obscure due to the absence of detailed molecular information on the composition of the Actinomyces cell surface and the lack of a facile genetic system to investigate the role of different components. Using a bioinformatics approach, we have identified the two fimbrial gene clusters in the unfinished genome of Actinomyces naeslundii MG-1. By biochemical and electron microscopy analysis, we have demonstrated that each gene cluster encodes a distinct fimbrial structure comprised of a fimbrial shaft protein and a minor subunit located largely at the tip region. Their assembly into fimbrial structures requires a specific transpeptidase, sortase, found in the gene cluster. Furthermore, we also have identified many putative surface proteins, a majority of which are predicted to participate in cell-cell interactions based on sequence features. We hypothesize that interactions of Actinomyces with host cell receptors and infectious partners may involve various fimbrial and non- fimbrial factors. Thus, the long term goal of this proposal is to delineate the mechanisms of assembly of the surface molecules of Actinomyces and to examine their role in bacterial infection. Using a combination of electron microscopy, mass spectrometry and molecular biology, we aim to determine the molecular architecture of Actinomyces fimbriae and the chemical nature of their protein cross-linkages. Genetic and biochemical experiments will be employed to define the enzymes, substrates and products of fimbrial assembly. Importantly, we will determine whether fimbrial and non-fimbrial factors contribute to the interactions of Actinomyces with host cells and other etiological components. The results generated should provide ample, powerful experimental systems for future studies on the biological processes of these organisms in their ecological niche. Our proposal aims to characterize surface structures of oral bacteria Actinomyces that may play an important role in plaque formation. The generated results will permit the development of inhibitors which block the assembly of surface proteins or fimbriae that may prove a useful strategy for preventing carries and periodontal diseases.